The overall goal of this application is to explore the involvement of non-classical MHC class Ib (class Ib) molecules in thymocyte differentiation during early development by a comparative approach in the Xenopus. Increasing evidence suggests that in the absence or suboptimal expression of 'classical'MHC class Ia (class Ia) as it occurs in neonates, class Ib molecules can generate survival signals to differentiating thymocytes. Not much is known about the physiological relevance of this alternative thymic selection process. It is hypothesized that in the absence (or suboptimal level) of class Ia expression early in development, thymic differentiation and T cell education critically depends on class Ib. Both the thymic dependence of T cells and the factors that play key roles in the regulation of T cell development are broadly conserved among jawed vertebrates. Therefore, the investigators propose to use the Xenopus and its immunocompetent but naturally class Ia-deficient larval stage as a model because it provides the advantage of having the early developing thymus of larvae externally visible, easily accessible, and amenable to experimental manipulation. Importantly, the hypothesis involving class Ib can be tested in the natural absence of class Ia, and the development of T cells can be studied in a biological context. Finally, the investigators have recently characterized a novel Xenopus nonclassical MHC class Ib (XNC) gene subfamily, XNC10, whose expression is tightly associated with the CD8 T-cell lineage during thymocyte differentiation since early in ontogeny. Therefore, they propose to test the hypothesis that class Ib genes are critically involved in thymic differentiation and T cell education during ontogeny of Xenopus. This will be achieved by the two following aims: 1. To determine the expression pattern of XNC10 and a selected set of immune genes during ontogeny: This will involve in situ hybridization as well as immunohistology and flow cytometry analysis for which they will generate new antibodies against XNC10 and other thymocytes markers (e.g., CD4, CD8[unreadable], TCR[unreadable], a);and 2. To assess the function of XNC10 in T cell differentiation and thymic education, which will involve in vivo knockdown by RNA interference using transgenesis to reveal the function of XNC10 in CD8 T cell development. This application will provide fundamental, and not phylogenetically restricted, insight on the relative contributions of class Ia versus class Ib-mediated thymocyte education pathways during ontogeny. Technologies developed and reagents obtained will be of high benefit for the scientific community and will considerably increase the potential of X. laevis as a non-mammalian alternative model. PUBLIC HEALTH RELEVANCE: The goal of this project is to better understand the possible role of certain MHC nonclassical class I molecules in assisting the development of fully mature T cells during early stage of life and ultimately helping their response against pathogens or tumors prone to escape the host's immune defenses. For this, the investigators will use an evolutionary approach that takes advantage of the frog X. laevis, which is a unique non-mammalian comparative model that permits easy access of the thymus early during development.